This invention relates to compositions and methods for administering Borrelia genospecies DNA encoding antigen(s) in vivo or in vitro. More particularly, this invention relates to compositions and methods for administering Borrelia genospecies DNA encoding an antigen or antigens, e.g., OspA (outer surface protein A) and/or OspB (outer surface protein B), and/or OspC (outer surface protein C), or fragments thereof such as fragments thereof containing at least one epitope of interest, for expression thereof, in vivo, ex vivo or in vitro.
Lyme disease is a multisystem illness, transmitted by ticks of the Ixodes ricinus complex. The spirochaete Borrelia burgdorferi sensu lato is the etiologic agent of Lyme disease, which is now the most common arthropod borne disease in the United States, and is endemic in Central Europe (Barbour and Fish 1993). More particularly, there are three genospecies of Borrelia associated with Lyme disease: Borrelia burgdorferi, Borrelia afzelii and Borrelia garinii. Borrelia burgdorferi is the etiologic agent of Lyme disease in North America, and some European Lyme disease is considered to be Borrelia burgdorferi sensu stricto. Borrelia afzelii and Borrelia garinii are the major cause of European Lyme disease and are considered Borrelia burgdorferi sensu lato.
Although Lyme disease is curable by antibiotic therapy in its early stages, if Lyme disease is allowed to progress, cardiac, neurological and joint abnormalities can arise. Investigations into the development of a human vaccine for Lyme disease are under way. The outer surface lipoprotein OspA of Borrelia burgdorferi is the current major candidate molecule for development of such a vaccine.
Recombinant OspA lipoprotein (rOspA) is known to elicit a protective immune response in mice against challenge by infectious B. burgdorferi (Fikrig et al., 1990; Erdile et al., 1993; U.S. Ser. No. 08/373,455). OspA is currently undergoing human field trials as a subcutaneously administered vaccine in the United States (Keller et al., 1994). Above-cited U.S. Pat. No. 5,688,512 relates to substantially pure OspA, vaccines including substantially pure OspA, and methods for inducing a protective immunological response against B. burgdorferi employing such vaccines, inter alia.
Above-cited applications Ser. No. 08/373,455 and PCT/US92/08697 relate to rOspA vaccines, especially lipidated rospA, and methods for expressing DNA encoding OspA. Above-cited applications Ser. No. 08/320,416 (now U.S. Pat. No. 5,582,990) and WO 90/04411 relate to DNA encoding OspA, the amino acid sequence of OspA, synthetic OspA, compositions containing OspA or synthetic OspA, and methods of using such compositions. Above cited application Ser. No. 08/137,175, filed Oct. 26, 1993 (now U.S. Pat. No. 5,777,095) relates to DNA coding for various OspAs and OspBs, OspAs and OspBs encoded by such DNA (including amino acid sequences therefor), and immunologically interesting fragments of OspAs and OspBs and DNA coding therefor.
In approximately half of the European isolates of B. burgdorferi, outer surface protein C (OspC) is the major surface antigen found on these spirochetes. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to B. burgdorferi strains expressing the homologous OspC protein (Preac-Mursic et al., INFECTION (1992) 20:342-349; Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926). Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospC gene of B. burgdorferi strain Pko and the OspC protein encoded thereby of 22 kDa molecular weight (termed xe2x80x9cpCxe2x80x9d therein). This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. As to DNA encoding OspC or recombinant OspC, reference is also made to WO96/05313 (Max-Planck Institute); Leuba-Garcia et al., Zentralbl Bakteriol. 287(4):475-84, 1998; Rauer et al., J. Clin. Microbiol. 36(4):857-61, 1998; Masuzawa et al., Clin. Diagn. Lab. Immunol. 4(1):60-63, 1997; Fukunaga et al. J. Clin. Microbiol. 33(9):2415-2420, 1995; Jauris-Heipke et al., J. Clin. Microbiol. 33(7):1860-66, 1995; Theisen et al., J. Bacteriol. 177(11):3036-3044, 1995; Stevenson et al. FEMS Microbiol. Lett. 124(3):367-72, 1994; and Padula et al., Infect. Immun. 61(12):5097-5105, 1993.
The other above-cited applications relate to DNA encoding other Borrelia antigens or other Osps, or to DNA encoding useful fragments of OspA or of other Osps, amino acid sequences thereof, compositions containing such fragments or other Osps, and methods for using such compositions; and, such DNA that can be used in the methods of Ser. No. 08/373,455 or PCT/US92/08697 to produce OspA, other Borrelia antigens or outer surface proteins (Osps), or fragments thereof, can be used in this invention. In regard to DNA useful in this invention, reference again made to V. Preac-Mursic et al., supra, W. S. Probert et al., supra, WO 91/09870, supra, as well as to all of the documents cited herein and also to Molecular Microbiology (1989), 3(4), 479-486, and PCT publications WO 93/04175, and WO 96/06165.
Alternative vaccination strategies are desirable as such provide alternative routes to administration or alternative routes to responses.
In particular, it is believed that heretofore the art has not taught or suggested administration to a eukaryotic cell in vitro or ex vivo, or to a mammalian hostxe2x80x94domesticated or wild or humanxe2x80x94susceptible to Lyme disease, of Borrelia genospecies DNA e.g., DNA encoding OspA and/or OspB, and/or OspC or expression thereof in vivo, especially as herein disclosed.
It is an object of the invention to provide methods and compositions for administering to a host, such as a mammalian host susceptible to Lyme Disease, Borrelia genospecies isolated and/or purified DNA encoding an antigen or antigens or a fragment or fragments thereof such as fragment or fragments containing at least one epitope of interest, e.g., isolated and/or purified DNA encoding an antigen or antigens or a fragment or fragments thereof such as fragment or fragments containing at least one epitope of interest from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii or combinations thereof, such as isolated and/or purified DNA encoding OspA, and/or OspB and/or OspC, or isolated and/or purified DNA encoding at least one epitope of OspA and/or OspB and/or OspC; for instance, DNA encoding Borrelia burgdorferi OspA and/or OspB and/or OspC. The compositions can include a carrier or diluent. The DNA is administered in a form to be expressed by the host, and preferably in an amount sufficient to induce a response such as a protective immune response; and, the DNA can be administered without any necessity of adding any immunogenicity-enhancing adjuvant.
Accordingly, the present invention provides Borrelia genospecies antigen or epitope DNA plasmids for expression by eukaryotic cells, compositions containing the plasmids, and methods for using the compositions and for using the products from the compositions.
The plasmid of the invention can comprise from upstream to downstream (5xe2x80x2 to 3xe2x80x2): DNA encoding a promoter for driving expression in eukaryotic cells, DNA encoding a leader peptide for enabling secretion of a prokaryotic protein sequence from a mammalian cell, Borrelia genospecies antigen or epitope DNA, and DNA encoding a terminator.
The DNA encoding a promoter for driving expression in eukaryotic cells can be a eukaryotic, e.g., mammalian, viral promoter, such as a herpes virus promoter. A human cytomegalovirus promoter is presently preferred. The human cytomegalovirus promoter can be an immediate early human cytomegalovirus promoter such as HCMV-IE. The plasmid can contain the HCMV-IE gene 5xe2x80x2 untranslated region (UTR) which includes Intron A. This sequence can be 3xe2x80x2 to the HCMV-IE promoter and 5xe2x80x2 to the portion of the chimeric 5xe2x80x2 UTR sequence and leader peptide (the UTR and leader peptide coding sequence can be derived from the DNA encoding the human tissue plasminogen activator, as discussed below).
The DNA encoding a leader peptide is for facilitating secretion of a prokaryotic protein sequence from a mammalian cell. This DNA can be any DNA encoding a suitable or similar leader peptide for the purpose of secretion from a mammalian cell, e.g., DNA encoding a eukaryotic leader peptide. For instance, the DNA encoding a leader peptide can be from DNA encoding a peptide hormone, i.e., a peptide hormone leader peptide, such as from a mammal, e.g., a human peptide hormone leader peptide. Specific examples of DNA encoding leader peptides suitable use in the invention include the DNA encoding the leader peptide of insulin (human, bovine, porcine, etc.), renin, Factor VIII, and tissue plasminogen activator.
DNA encoding human tissue plasminogen activator (TPA) leader is presently preferred. The DNA encoding TPA is derived from the TPA gene and encodes a portion of the 5xe2x80x2 UTR and leader peptide from the gene. TPA DNA having a portion of the 5xe2x80x2 UTR and leader peptide can even increase eukaryotic cell expression. Without wishing to necessarily be bound by any one particular theory, increased expression can be due to the 5xe2x80x2 UTR.
The Borrelia genospecies antigen or epitope DNA is preferably without the natural leader sequence. The Borrelia genospecies antigen or epitope DNA can preferably encode at least one antigen selected from OspA, OspB, OspC, OspD, other Osps, and other antigens (see documents cited herein). Similarly, the Borrelia genospecies antigen or epitope DNA can preferably encode at least one epitope of interest from an antigen selected from OspA, OspB, OspC, OspD, other osps, and other antigens. DNA without the natural leader sequence encoding OspA and/or OspB and/or OspC and/or an epitope of interest from OspA and/or OspB and/or OspC is presently preferred. The DNA can be from Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii or from any combination thereof; with Borrelia burdorferi presently preferred.
The terminator can be any suitable terminator sequence for use in eukaryotic cells; for instance, a terminator sequence from a mammalian peptide hormone. The Bovine Growth Hormone (BGH) terminator is presently preferred.
The plasmid of the invention can contain genes for a fusion protein of the activator (e.g., TPA) and the Borrelia antigen (e.g., OspA, OspB, OspC) or at least an epitope thereof.
The invention comprehends compositions comprising the plasmid and a carrier. The carrier can be any suitable carrier which does not disrupt the DNA or the function of the plasmid. Saline is a presently preferred carrier.
The invention further comprehends methods of using the compositions, e.g., administering the composition to a host susceptible to Lyme disease to elicit a response or express an antigen. The response can be protective. The response can be simply an antibody response, and the method can further include recovering antibodies and/or antibody-producing cells (with the cells further useful for producing hybridoma cells and monoclonal antibodies, e.g., by fusing the antibody-producing cells to produce hybridoma cells, and then obtaining monoclonal antibodies from expression by the hybridoma cells). The host can be a mammal, such as a human.
Still further, the invention provides methods for expressing an antigen or epitope or preparing an antigen or epitope in vitro comprising transfecting eukaryotic cells with an inventive plasmid. The method can further comprise recovering the antigen or epitope from the cells.
Methods for preparing the plasmid by ligating the respective pieces of DNA (DNA molecules) from which it is comprised, and methods for using the antibodies and antigens or epitopes, as well as the antigens or epitopes and antibodies themselves, are contemplated by the present invention.
Other objects and embodiments are disclosed or are obvious from the following Detailed Description.